Identification of a roseoflavin exporter in the roseoflavin producer Streptomyces davaonensis

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The bacterium Streptomyces davaonensis produces the antibiotic roseoflavin in the stationary phase of growth. Roseoflavin is a structural riboflavin (vitamin B2) analog and acts as an antimetabolite. The Mack group was able to show that roseoflavin negatively affects FMN riboswitches as well as flavoproteins and thus the antibiotic has multiple cellular targets. The Mack group also has carried out fundamental work with regard to the biosynthetic pathway of roseoflavin. One important open question is at present how roseoflavin is exported from the producer cell. Our preliminary experiments clearly show that a yet unknown transporter is responsible for roseoflavin export as roseoflavin is found in the supernatant of cultures of the producer cell. Such an exporter protein must be highly specific to avoid export of essential riboflavin. Export of roseoflavin is important for the producer, otherwise the compound would not be able to inhibit growth of competing microorganisms in a natural environment. The objective of the present grant application is the identification of the roseoflavin exporter in S. davaonensis.Five different work packages describe experiments which should lead to the identification of the roseoflavin exporter. It is unclear at present whether a single protein or whether multiple proteins form this unique exporter. We plan to carry out systematic deletion experiments and co-expression experiments in different bacterial backgrounds to identify the roseoflavin exporter. In case these experiments fail we plan to carry out protein-protein interaction experiments. The hypothesis thereby is that the last enzyme of roseoflavin biosynthesis, RosA, which binds its reaction product roseoflavin very tightly, interacts with a membrane bound exporter. It is also possible that RosA interacts with a binding protein which then brings roseoflavin to the elusive exporter. Employing protein-protein interaction studies binding partners of RosA can be identified, which may help to identify components of the roseoflavin export system. In principle uptake systems – especially for riboflavin - are well described in bacteria. In contrast, only very few exporters are known which makes the present grant application especially interesting.